Van Poelvoorde Laura A E, Picalausa Corinne, Gobbo Andrea, Verhaegen Bavo, Lesenfants Marie, Herman Philippe, Van Hoorde Koenraad, Roosens Nancy H C
Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, Belgium.
Foodborne Pathogens, Sciensano, 1050 Brussels, Belgium.
Microorganisms. 2023 Mar 12;11(3):729. doi: 10.3390/microorganisms11030729.
Wastewater-based surveillance can be used as a complementary method to other SARS-CoV-2 surveillance systems. It allows the emergence and spread of infections and SARS-CoV-2 variants to be monitored in time and place. This study presents an RT-ddPCR method that targets the T19I amino acid mutation in the spike protein of the SARS-CoV-2 genomes, which is specific to the BA.2 variant (omicron). The T19I assay was evaluated both in silico and in vitro for its inclusivity, sensitivity, and specificity. Moreover, wastewater samples were used as a proof of concept to monitor and quantify the emergence of the BA.2 variant from January until May 2022 in the Brussels-Capital Region which covers a population of more than 1.2 million inhabitants. The in silico analysis showed that more than 99% of the BA.2 genomes could be characterized using the T19I assay. Subsequently, the sensitivity and specificity of the T19I assay were successfully experimentally evaluated. Thanks to our specific method design, the positive signal from the mutant probe and wild-type probe of the T19I assay was measured and the proportion of genomes with the T19I mutation, characteristic of the BA.2 mutant, compared to the entire SARS-CoV-2 population was calculated. The applicability of the proposed RT-ddPCR method was evaluated to monitor and quantify the emergence of the BA.2 variant over time. To validate this assay as a proof of concept, the measurement of the proportion of a specific circulating variant with genomes containing the T19I mutation in comparison to the total viral population was carried out in wastewater samples from wastewater treatment plants in the Brussels-Capital Region in the winter and spring of 2022. This emergence and proportional increase in BA.2 genomes correspond to what was observed in the surveillance using respiratory samples; however, the emergence was observed slightly earlier, which suggests that wastewater sampling could be an early warning system and could be an interesting alternative to extensive human testing.
基于废水的监测可作为其他新冠病毒监测系统的补充方法。它能及时、准确地监测感染情况以及新冠病毒变体的出现和传播。本研究提出了一种逆转录数字滴度聚合酶链反应（RT-ddPCR）方法，该方法针对新冠病毒基因组刺突蛋白中的T19I氨基酸突变，此突变是奥密克戎BA.2变体所特有的。对T19I检测法的包容性、敏感性和特异性进行了计算机模拟和体外评估。此外，以废水样本作为概念验证，监测并量化了2022年1月至5月布鲁塞尔首都大区BA.2变体的出现情况，该地区人口超过120万。计算机模拟分析表明，使用T19I检测法可对99%以上的BA.2基因组进行特征分析。随后，成功通过实验评估了T19I检测法的敏感性和特异性。由于我们特定的方法设计，测量了T19I检测法中突变探针和野生型探针的阳性信号，并计算了具有BA.2突变特征的T19I突变基因组在整个新冠病毒群体中所占的比例。评估了所提出的RT-ddPCR方法在监测和量化BA.2变体随时间出现情况方面的适用性。为了验证该检测法作为概念验证的有效性，于2022年冬春季节，在布鲁塞尔首都大区污水处理厂的废水样本中，测量了含有T19I突变的基因组在特定循环变体中所占比例相对于总病毒群体的比例。BA.2基因组的这种出现情况和比例增加与呼吸道样本监测中观察到的情况一致；然而，BA.2变体的出现略早，这表明废水采样可能是一个早期预警系统，并且可能是广泛人群检测的一个有趣替代方案。
